Nearly one-third of the eukaryotic proteome traverses the cellular secretory pathway [Imperiali, Acc. Chem. Res. 30, 452-459 (1997)]. Many of these proteins are co-translationally N-glycosylated at Asn residues within the conserved Asn1-Yyy2-Thr3/Ser3 sequon, where Yyy is any amino acid residue other than proline and is located at position 2 between an asparagine (Asn) at the amino-terminal end of the sequon and a threonine or serine (Thr/Ser) at the carboxy-terminal end of the sequon. N-Glycosylation can increase the stability of proteins, however the molecular basis for this is enhanced stability is incompletely understood.
As the ribosome inserts polypeptide chains into the endoplasmic reticulum (ER), the enzyme oligosaccharyl transferase (OST) attaches the highly conserved Glc3Man9GlcNAc2 (where Glc is glucose, Man is mannose, and GlcNAc is N-acetylglucosamine) glycan (oligosaccharide) en bloc to the N atom of the Asn side chain in a subset of Asn-Xxx-Thr/Ser sequons [Kornfeld et al., Annu Rev Biochem 54, 631-664 (1985); and Kelleher et al., Glycobiology 16:47 R-62R (2006)]. N-linked glycans have important extrinsic effects on folding in the ER by allowing glycoproteins to enter the calnexin/calreticulin (CNX/CRT) folding/degradation pathway [Molinari, Nat Chem Biol 3, 313-320 (2007); Helenius et al., Science 291, 2364-2369 (2001)]. N-glycans can also have intrinsic effects on protein folding by enhancing protein folding efficiency in cells, even when the CNX/CRT pathway is absent [Banerjee et al., Proc Natl Acad Sci USA 104, 11676-11681 (2007); Trombetta, Glycobiology 13, 77R-91R (2003)] or when the N-glycan does not allow CNX/CRT interactions [Stanley et al., FASEB J 9, 1436-1444 (1995)], consistent with reports that N-glycans stabilize protein structure, accelerate folding, and reduce aggregation in vitro [Wormald et al., Structure with Folding & Design 7, R155-R160 (1999); Jitsuhara et al., J Biochem 132, 803-811 (2002); Mitra et al., Trends in Biochemical Sciences 31, 156-163 (2006)].
The increased use of protein therapeutics has made issues such as stabilized polypeptide structure, accelerated folding, and reduced aggregation of paramount importance to the pharmaceutical industry [Li et al., Curr Opin Biotechnol 20, 678-684 (2009); Sinclair et al., J Pharm Sci 94, 1626-1635 (2005); Sola et al., BioDrugs 24, 9-21 (2010); Walsh et al., Nat Biotechnol 24, 1241-1252 (2006)]. The therapeutic benefits of N-glycosylation are exemplified in darbepoetin alfa (an erythropoietin variant with two additional N-glycans) [Egrie et al., Exp Hematol 31, 290-299 (2003), interferon β [Runkel et al., Pharm Res 15, 641-649 (1998)], and follicle stimulating hormone [Perlman et al., J Clin Endocrinol Metab 88, 3227-3235 (2003)].
A number of types of tight turns within secondary protein or polypeptide sequences have described in the literature. These structures are referred to as a δ-turn that encompasses two amino acid residues, a γ-turn that involves three residues, a β-turn that involves four amino acid residues, an α-turn that involves five residues and a π-turn that involves six residues. [Chou, Anal Biochem 286, 1-16 (2000).]
A β-turn or reverse turn contains a sequence of four consecutive amino acid residues that are designated i, i+1, i+2 and i+3, in the direction from N-terminus toward C-terminus of the polypeptide. The five residues of an α-turn are designated i, i+1, i+2, i+3 and i+4. Most, but not all reverse turns and α-turns contain a hydrogen bond between the first and fourth or first and fifth residues, respectively, in which the residue designated i contains a peptide bond (peptidyl) carbonyl group (>C═O), whereas the fourth residue, i+3, or the fifth residue, i+4, contains the peptidyl —NH— group whose hydrogen is hydrogen-bonded to the carbonyl oxygen of the i residue. Residues bonded to the amino group of the i residue (toward the amino-terminus from the i residue) are designated i−1, i−2, i−3, etc.
Another way to define a reverse turn and an α-turn motif is by the close approach, less than 7 Å, of Cα atoms (alpha-carbon atoms) of the residues of the motif. Thus, one can define a β-turn and an α-turn by the close approach of Ca atoms of residues I and i+3 or i and i+4, respectively. [Chou, Anal Biochem 286, 1-16 (2000).] This distance implies a particular geometry of the corresponding backbone, which turns back on itself or, more generally, that corresponds to a change of direction, and that the residue side chains are on the same side of the backbone chain.
The β-turns are usually described as orienting structure because they orient α-helices, and β-sheets, indirectly defining the topology of proteins. They are one of the most abundant secondary structures.
Several types of reverse turns have been identified and are designated types I, I′, II, III, IV, V and VI. Types I and II are the most common reverse turns, the essential difference between them being the orientation of the peptide bond between residues at i+1 and i+2. The i+2 residue of the type II turn can substantially only be occupied by glycine because of steric interference of the carbonyl group of the i+1 residue.
It was recently shown that naturally occurring N-glycosylation at a single Asn residue comprising a reverse turn within the adhesion domain of human glycoprotein CD2 (HsCD2ad) stabilizes the protein by −3.1 kcal mol−1, makes folding four times faster, and makes unfolding 50 times slower in vitro [Hanson et al., Proc Natl Acad Sci USA 106, 3131-3136 (2009)]. However, introducing N-glycans into proteins that are not normally glycosylated (naïve proteins) has previously rarely led to substantially improved folding energetics [Hackenberger et al., J Am Chem Soc 127, 12882-12889 (2005); Wang et al., Biochemistry 35, 7299-7307 (1996); Elliott et al., J Biol Chem 279, 16854-16862 (2004)].
The present inventors and co-workers recently showed that glycosylation of an Asn residue within the sequence Aro-(Xxx)n-(Zzz)p-Asn-Yyy-Thr/Ser, where Aro is an aromatic amino acid residue such as histidine, phenylalanine, tyrosine or tryptophan, n is zero, 1, 2, 3 or 4, Xxx is an amino acid residue other than an aromatic residue, p is zero or one, Zzz is any amino acid residue, Asn is asparagine, Yyy is any amino acid residue other than proline, Thr/Ser is one or the other of the amino acid residues threonine and serine, stabilizes the glycosylation-naïve rat CD2 adhesion domain (RnCD2ad) and human muscle acylphosphatase (AcyP2) by about −2 kcal mol−1, provided that Asn is located at the i+2 position of a type I β-turn with a G1 β-bulge using the terminology of Sibanda et al., J Mol Biol 206(4), 759-777 (1989); Richardson, Adv Protein Chem 34, 167-339 (1981), hereafter called a type I β-bulge turn [Culyba et al., Science 331, 571-575 (2011); Application Ser. No. 61/380,967, filed 8 Sep. 2010].
Published structural data [Wyss et al., Science 269, 1273-1278 (1995)] from the human ortholog of RnCD2ad (HsCD2ad, FIG. 1A) suggest that placement of an N-glycan at i+2 in the type I β-bulge turn context permits the α-face of GlcNAc1 of the N-glycan to engage in stabilizing hydrophobic interactions with the aromatic ring of Phe at the i position, and the side-chain methyl group of Thr at the i+4 position {a stabilizing C—H/n interaction may also play a role [Laughrey et al., J Am Chem Soc 130(44), 14625-14633 (2008)]}.
Thus, it is hypothesized that the substantial energetic benefits of glycosylating a protein such as HsCD2ad depend on both the reverse turn context of the glycosylation site and the surrounding amino acid sequence. Some results showing the correctness of this hypothesis as applied to therapeutic polypeptides are shown and discussed hereinafter.